The Na/K-ATPase (NKA) is the main Na extrusion pathway and is essential in [Na]i regulation. In heart, [Na]i critically modulates [Ca]i, contractiity and triggered arrhythmias via Na/Ca exchange. Thus, NKA regulation is functionally important, and our prior work on this project has made several seminal observations. First, phospholemman (PLM) regulates NKA similar to the way phospholamban (PLB) regulates SR Ca-ATPase (SERCA). That is, PLM binds to and inhibits NKA by reducing the [Na]i-affinity, but PLM phosphorylation relieves this inhibition to increase NKA function. Second, PLM phosphorylation and the consequent NKA stimulation is integral to the sympathetic fight or flight response (tempering the rise in [Na]i and cellular Ca load, and limiting Ca overload-induced arrhythmias). Third, PLM physically associates with NKA, but in bimolecular FRET measurements, PLM phosphorylation alters the PLM-NKA interaction. Fourth, PLM also forms homo-oligomers, which are favored by PLM phosphorylation. Fifth, we identified sites on both NKA (E960) and PLM (F28) that are essential for the regulatory interaction. Sixth, that block of NKA-?2 (vs. ?1) has a preferential inotropic effect. Here we will further decipher NKA-PLM regulation in heart, using FRET, [Na]i and [Ca]i measurements, patch-clamp and molecular biology in myocytes and HEK 293 cells and structural modeling. Aim 1 will identify PLM (& NKA) sites that are critical for PLM-PLM interaction, and also elucidate the molecular structure of PLM multimers (which we suspect are tetramers). We will also test whether PLM-PLM disruption promotes PLM-NKA interaction and pump inhibition. Aim 2 will use novel NKA mutants to test the molecular basis of and functional effects of NKA-NKA dimerization on pump activity, PLM interaction and signaling. We will also examine how ouabain disrupts these natural dimers. Aim 3 will test whether membrane domains from PLB or FXYD4 (which can stimulate NKA), can alter PLM effects on NKA in PLM/PLB or PLM/FXYD4 chimeras. We will also test if novel selective NKA-?2 inhibitors might be an improved modern-day digitalis therapy. These studies will greatly enrich our mechanistic understanding of NKA regulation in heart and explore novel therapeutic strategies.